Hybridoma antibody which inhibits Interleukin 2 activity

ABSTRACT

A process for producing anti-IL-2 antibody from hybridoma cells generated by fusing activated, IL-2 immunized, murine lymphocyte cells with neoplastic murine myeloma cells. Fusion is accomplished by mixing the two cell lines together in the presence of a fusing agent. After fusion, the hybridoma cells are cultured in vitro in a supplemented tissue culture medium to thereby produce anti-IL-2 antibody. Also, the hybridoma cells are cloned by a limiting dilution procedure to isolate even more potent sources of anti-IL-2 antibody. Anti-IL-2 antibody is then purified from either tissue culture medium conditioned by hybridoma cells, or from peritoneal ascites of mice challenged with hybridoma cells.

DESCRIPTION CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of my copending U.S. patent applicationSer. No. 258,601, now U.S. Pat. No. 4,411,993, filed Apr. 29, 1981.

TECHNICAL FIELD

The present invention relates to an antibody which inhibits Interleukin2 (hereafter "IL-2") (formerly known in the literature as "T cell growthfactor" or "TCGF") activity and a process for preparing same, and moreparticularly to a process for producing murine anti-IL-2 antibody fromhybrid cells formed by the fusing of murine myeloma cells with murineanti-IL-2 antibody producing B-lymphocytes.

BACKGROUND ART

IL-2 is a soluble protein which has been found to influencecell-mediated immune responses in mammals; and in the past, has beenproduced by stimulating mouse, rat or human lymphocytes with a T cellmitogen. The immune responses which have been attributed to IL-2,include: (i) enhancement of thymocyte mitogenesis; (ii) production ofalloantigen-directed cytotoxic T lymphocytes in spleen cell thymocyteand athymic nude mouse spleen cell cultures; (iii) promotion of longterm in vitro proliferation of antigen specific helper or killer T celllines; and (iv) promotion of anti-erythrocyte (red blood cell) plaqueforming cell responses in nude mouse spleen cell cultures stimulatedwith sheep red blood cells.

From its ability to influence T cell dependent immune responses invitro, it is thought that IL-2 plays a significant role in cell mediatedimmunity in vivo. The study of the in vivo capabilities of IL-2 has beenhindered by the lack of reagents which are capable of detecting thepresence of IL-2 serologically, as opposed to having to monitor itsbiological effect. Moreover, further examination not only of themolecular properties of this immunoregulatory molecule, but also of thebiochemical comparisons between IL-2 and other lymphokine activitieswould benefit from the development of antibodies which react with IL-2.Accordingly, a principal object of the present invention is to develop aprocess for preparing an antibody which inhibits IL-2 activity which maybe used to detect serologically its presence.

DISCLOSURE OF THE INVENTION

The present invention relates to a process for producing anti-IL-2antibody from murine hybridoma cells. The process includes fusinganti-IL-2 antibody producing murine B-lymphocytes with murine myelomacells and then cultivating the resulting hybrid cells by eitherculturing the hybrid cells in vitro in a selective medium to therebyliberate an anti-IL-2 antibody containing supernate or growing thehybrid cells in vivo by intraperitoneal injection of mice with thehybrid cells to thereby generate high concentrations of anti-IL-2antibody. Prior to the fusion process, murine splenocytes, whichpreviously have been immunized in vivo to rat IL-2, are activated byculturing the cells in vitro in a tissue culture medium containing a Bcell mitogen. In the fusion process, the stimulated anti-IL-2 antibodyproducing cells are mixed with myeloma murine driver cells and then afusing agent is added thereto. When cultivating the resulting fusedcells by culturing, the cell mixture is cultured in a mediumsupplemented with selective compounds which prevent the growth ofunfused driver cells. The process of the present invention results inhybrid cells which exhibit both the anti-IL-2 antibody generatingcapacity of the activated B-lymphocytes and the immortality of thedrug-marked myeloma cells.

The process of the present invention also includes cloning the murineanti-IL-2 antibody producing hybrid cells by suspending single cellsamples of the hybrid cells in vitro in a tissue culture medium. Afterthe single cell cultures have grown to appropriate densities, the cellsare resuspended in culture medium to continuously generate anti-IL-2antibody.

The above-described hybridizing and anti-IL-2 antibody productionprocess has been employed in conjunction with splenocytes harvested fromBALB/c mice together with SP2/0AG14 hereafter "SP2") myeloma drivercells also from BALB/c mice. The anti-IL-2 antibody producing portion ofthe present invention has been carried out with hybrid cells generatedby fusion of these two cell lines and by clones of such hybrid cells.Applicant has identified a particular clonal hybrid cell line,designated as 4E12B2D10, which is capable of significantly neutralizingIL-2 activity from murine, human and rat sources.

Tests conducted on the supernate generated by the hybrid cell linesproduced by the present invention suggest that the anti-IL-2 antibodycontained therein is a monoclonal B cell hybridoma whose immunoglobulinG product appears to be directed against a determinant present onmolecules of human, murine and rat IL-2. This conclusion is based on thefinding that passage of cloned hybrid cell culture supernatants througha Protein A-coupled Sepharose column yielded purified immunoglobulin Gfractions which inhibited mouse, rat and human IL-2 activity. Secondly,hybridoma-derived IgG, in concert with lyophilized Staphylococcusaureus, was capable of precipitating both "cold" and intrinsicallylabeled IL-2 activity. Moreover, Sepharose conjugated with purified IgGfractions provided an extremely reactive IL-2 absorbtion matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of typical embodiments of the present invention will bedescribed in connection with the accompanying drawings, in which:

FIG. 1 shows the results of neutralization ( ) and precipitating ( ○ )screening trials monitoring the isolation of the parent cell line,designated as "4", and the 4E12, 4E12B2 and 4E12B2D10 clonal cell lines;

FIG. 2 shows the results of neutralization (A) and precipitating (B)screening cultures detailing the inhibitory effects (on IL-2 dependentCTLL cell proliferation) of supernates harvested from 4E12B2D10 ( )5G8H5C6 ( ) and 2D4A7 ( ) hybrid cell cultures;

FIG. 3 shows Igsorb-precipitation screening cultures monitoring theanti-IL-2 effects of HAT medium ( ○ ), SP2 conditioned medium ( ),supernate harvested from the clonal hybridoma 4E12B2D10 cell line ( ).The capacity of the 4E12B2D10 supernate to inhibit IL-2 dependent CTLLcell [³ H]Tdr incorporation was lost by passage of the supernate over a3 ml column of Protein A conjugated Sepharose ( ). However, enhancedanti-IL-2 inhibitory activity could be recovered from the column bystandard acid elution using glycine-HCl (pH3) buffer and subsequentdialysis (□);

FIG. 4 shows the results of CTLL proliferation assays testing IL-2activity remaining in the standard preparation of rat spleen cellcondition medium (3 U/ml) either before ( ) or following Igsorbprecipitation in the presence of Click's medium (Δ), SP2 conditionmedium ( ○ ), HAT medium ( ), or Protein A Sepharose purified 4E12B2D10IgG ( );

FIG. 5 shows IL-2 activity .[.(○).]. .Iadd.( ) .Iaddend.and cpm of ³H-label .[.(Δ).]. .Iadd.( ) .Iaddend.in Sephadex G100 fractions ofbiosynthetically radio-labelled LBRM-33 cell derived IL-2 preparations;and,

FIG. 6 shows IL-2 activity present in 3 U/ml sample of rat spleen cellconditioned medium both prior to ( ) and the following passage over aBSA conjugated .[.Sepherose.]. .Iadd.Sepharose .Iaddend.column ( ) or anidentical column to which 4E12B2D10 IgG had been coupled ( ).

BEST MODE OF THE INVENTION Outline of Process

In accordance with the present invention, murine B lymphocytes, whichpreviously have been immunized in vivo with rat IL-2, are activated byculturing the cells in vitro in a tissue culture medium which alsocontains a B cell mitogen. The mitogen stimulated lymphocyte cells arefused with malignant, murine myeloma cells by mixing the two differenttypes of cells together and then pelleting the mixture. The cell pelletis then resuspended in a tissue culture medium which also contains afusing agent. Thereafter, the cell solution is pelleted again andresuspended in another protein containing medium which is supplementedwith various additives, feeder cells and selective suppressing agentsprecluding the growth of unfused myeloma cells thereby liberating ananti-IL-2 antibody producing hybrid cell. Alternatively, anti-IL-2antibody may be expanded by injecting the hybridoma cells from the cellpellet into the peritoneal cavities of mice and thereafter collectingthe interperitoneal ascites which contain high concentrations ofanti-IL-2 antibody (see Example 3 below).

The present invention also concerns identifying potent anti-IL-2antibody producing cell lines by cloning hybrid cell lines found toproduce this antibody .[.contitutively.]. .Iadd.constitutively.Iaddend..Cloning is accomplished by a limiting dilution procedure whereinanti-IL-2 antibody producing hybrid cells are individually cultured invitro in culture medium containing feeder cells and selectivesuppressing agents which prevent the growth of unfused myeloma cells.

In the process of the present invention, BALB/c female mice have beenutilized as a source of anti-IL-2 antibody producing B-lymphocytes.Activated spleen cells from these IL-2 immunized mice have been fusedwith the SP2 murine myeloma cell line, also derived from BALB/c mice, toproduce several hybrid cell lines capable of constitutive anti-IL-2antibody production (see FIG. 1). Cloning of one of the most potent IL-2inhibitor cell lines, denominated as 4E12, has resulted in theidentification of various clonal hybrid cell lines which are capable ofproducing anti-IL-2 antibody, including a cell line labeled as 4E12B2.An even more potent antibody source, designated as 4E12B2D10, has beenisolated by subcloning the 4E12B2 clonal cell line. Samples of the 4E12,4E12B2 and 4E12B2D10 are on deposit with the American Type CultureCollection, 12361 Parklawn Drive, Rockville, Md. 20852, under accessionnumbers HB8093, HB8092, and HB8091, respectively.

The effectiveness of the anti-IL-2 antibody produced by the parent andclonal cell lines, set forth above and in FIG. 1, may be ascertained bytesting the capacity of the antibody to inhibit the ability of IL-2 toinduce effector T cell replication. In the test procedure, the supernateproduced by the hybrid cells of the present invention are mixed withcytotoxic T lymphocytes (hereafter "CTLL") in a supplemented tissueculture medium containing IL-2. The CTLL cells which proliferate underthe stimulation of IL-2 will incorporate tritiated thymidine (hereafter"[³ H]Tdr") whereas cells which are cultured in the absence of IL-2 willincorporate only control levels of [³ H]Tdr. Thus, in FIG. 1, the lowerthe level of [³ H]Tdr incorporation, the more effectively the antibodyproduced by a particular cell line inhibits IL-2 activity.

FIG. 1 shows the results of neutralization () and precipitating ( ○ )screening trials monitoring the isolation of the 4E12B2D10 hybridoma. Inthe initial plate "4" culture (panel A) only microwell culture 4E12produced supernate which significantly inhibited IL-2 dependent CTLLcell [³ H]Tdr incorporation. Subsequent limiting dilution cloning gaverise to the hybrid cell lines screened in panel B of which the linedesignated 4E12B2 produced the most inhibitory supernate. Additionalsubcloning and screening results (panel C) show that all 4E12B2 daughterclones produce supernates which markedly inhibit IL-2 dependentproliferation in neutralizing the precipitating screening cultures.

As outlined above, in the anti-IL-2 antibody production process of thepresent invention, prior to fusion, BALB/c mice are immunized with IL-2and antibody producing B-lymphocytes harvested from their spleens.Preferably the immunization is conducted periodically, and in variousamounts to induce in vivo generation of anti-IL-2 activity. Ideally theimmunizations are conducted weekly for four weeks in 3000 unit doses.Rather than utilizing IL-2 singularly, it may be mixed with complete orincomplete Freunds adjuvant. Also, rather than injecting the entirevolume of IL-2 in one body location, preferably on each occasionmultiple injections are placed about the body of the mice, for instancein the hind legs and in the peritoneal cavity.

Also, according to the present invention, prior to fusion, spleen cellsfrom the IL-2 .[.ummunized.]. .Iadd.immunized .Iaddend.mice are removedand single cell suspensions prepared therefrom. The splenocytes arecultured in a tissue culture medium supplemented with various additives.A B cell mitogen is added to the culture medium to activate the "immune"spleen cells, prior to fusion.

In the process of the present invention IL-2 is employed to inducemurine splenocytes to produce anti-IL-2 antibody. As discussed morefully below, IL-2 is also employed in a process for screening hybridomasupernate samples for their capacity to inhibit IL-2 activity. IL-2 forthese uses may be produced by culturing murine splenocytes, orparticular murine thymoma (LBRM-33) or human leukemia (Jurkat-FHCRC)cells in vitro in a protein containing medium supplemented with variousadditives. A T cell mitogen is added to the culture medium to stimulateproduction of supernate which contains IL-2. After a period of time, thesupernate is collected and processed to purify the IL-2 into a moreconcentrated form, for instance, by sequential ammonium .[.sulphate.]..Iadd.sulfate .Iaddend.precipitation, gel exclusion chromatography, ionexchange chromatography, and preparative flat-bed iso-electricalfocusing.

Different types of murine myeloma cells may be fused with the activatedantibody producing splenocytes to thereby infuse the hybrid cells withthe ability to proliferate after fusion. One such driver drug-markedmyeloma cell line is the SP2 myeloma cell derived from the BALB/c mouse.Additional driver cell lines include NS-1, P3, XC3, Ag8 and otherdrug-marked BALB/c mouse myeloma cell lines.

The fusion step of the present invention includes combining drivermyeloma cells with activated anti-IL-2 antibody producing cells by firstmixing the cells and then pelleting them by centrifugation. Thereafter,the cell pellet is suspended in a slightly alkaline tissue culturemedium which also contains a fusing agent that facilitates fusion of thetwo different types of cells. Fusing agents may include various types ofcondensation polymers of ethylene oxide and water, such as polyethyleneglycol (hereinafter "PEG") 1500. Other fusing agents includedeoxyribonucleic acid (hereinafter "DNA") transforming viruses, such asSendai virus or the fusion protein obtained therefrom. For optimumfusion, the quantity and concentration of the fusing agent must becontrolled. For instance, if PEG 1500 is used to fuse immunized BALB/cmice splenocytes with SP2 myeloma cells, this fusing agent shouldcomprise about 40% (weight/volume). However, the volume of PEG 1500 mayrange from 0.5 to 3 milliliters and the concentration of PEG 1500 mayvary from 35% to 60% weight/volume of culture medium.

The process of the present invention involves several culturingprocedures, including during: (i) production of IL-2 for use in (a)immunizing animals to allow for in vivo generation of the anti-IL-2antibody producing cells, and (b) screening hybridoma supernates fortheir capacity to inhibit IL-2 activity; (ii) initial activation of theparent, anti-IL-2 antibody producer cells with a B cell mitogen prior tohybridization; (iii) fusion of anti-IL-2 antibody producer cells withdrug-sensitive myeloma cells; (iv) cloning and subcloning of hybridomaantibody producer cells; (v) anti-IL-2 antibody production by parent andclonal hybrid cells; and (vi) screening, concentrating and purifying theanti-IL-2 antibody present in the hybridoma supernates. Various types ofappropriate cell culturing media, which have been previously found tofoster growth of murine lymphocyte cells, may be utilized in thesedifferent culturing steps. The culture media include Roswell ParkMemorial Institute (hereafter "RPMI") 1640 medium, Click's medium, andDulbecco's Modified Eagle's medium (hereafter "DMEM").

In the production of anti-IL-2 antibody containing supernates from theparent and clonal hybridoma cells, the culture media may be supplementedwith various individual additives or combination of additives, includingfetal calf serum (hereafter "FCS"), which has been heat-inactivated by,for example, applying heat at 56° C. for approximately 30 minutes. Thequantity of FCS added may be from 5 to 20% of the total culture volume.Another additive is penicillin at a concentration range of approximately25 to 250 units per milliliter, and preferably approximately 50 unitsper milliliter. Streptomycin also may be utilized as an additive in apreferred concentration range of from 20 to 250 micrograms permilliliter of total culture volume, and ideally approximately 50micrograms per milliliter. Further additives include: (i) sodiumpyruvate in a concentration range of approximately 10 to 150 millimolarand ideally approximately 100 millimolar; (ii) N-2-hydroxypiperazine-XI¹-2-ethene-sulfonic acid (hereafter "Hepe's") buffer in a preferredconcentration of from 10 to 60 millimolar, and ideally approximately 25millimolar; and (iii) fresh L-glutamine in a preferred concentrationrange of approximately 150 to 500 micrograms per milliliter, with anideal concentration of approximately 300 micrograms per milliliter. Inaddition, NaHCO₃ in concentration range of 1 to 30 millimolar andideally about 16 millimolar may be added to the culture media.

As discussed above, in the anti-IL-2 antibody production process of thepresent invention, prior to fusion, the antibody producer cells, such asthe immunized BALB/c splenocytes, are activated with a B cell mitogen.Also, T cell mitogens are utilized in the culturing process to preparerat IL-2 for use in immunizing the anti-IL-2 antibody producing cellline, such as the BALB/c mouse, and to prepare murine and human IL-2 foruse in testing the ability of the anti-IL-2 antibody of the presentinvention to inhibit anti-IL-2 activity, discussed more fully below. Tcell mitogens may include various commercially available standard plantglycoproteins, such as phytohemagglutinin (hereafter "PHA"),concanavalin A (hereafter "Con A") and pokeweed mitogen (hereafter"PKM"). Although different concentrations of a particular mitogen may beemployed, applicant has found that if PHA is used, a concentration ofapproximately 1% by volume is sufficient to stimulate some murine(LBRM-33) and human malignant (Jurkat-FHCRC) cell lines into IL-2production. If Con A is employed to stimulate mouse or rat IL-2production, approximately 5 micrograms per milliliter of culture mediumshould be utilized.

In the stimulation of harvested murine spleens prior to fusion, thesplenocytes may be activated with the B cell mitogen E. Colilipopolysacchride (hereafter "LPS") or with PKM. If LPS is employed,ideally it can be added in a concentration of about 20-100 microgramsper milliliter of culture medium.

During the process of culturing the hybridoma cells after fusion, parentmyeloma cell growth inhibitors or suppressants such as hypoxanthine,aminopterin and thymidine (hereafter collectively referred to as "HAT")may be added to the culture medium to prevent unfused myeloma cells fromproliferating. Hypoxanthine may be added in an .[˜]. .Iadd.amount.Iaddend.of 10 to 20 milligrams per liter of culture medium, andpreferably about 13.6 milligrams per liter. Also, the concentration ofaminopterin may be from 0.1 to 0.2 milligrams per liter of culturemedium, and preferably about 0.176 milligrams per liter; and theconcentration of thymidine may be from 3.0 to 4.5 milligrams per literof culture medium and ideally about 3.88 milligrams per liter.

During the process of culturing the fused cells and the clones thereof,feeder cells may be added to the culture medium to induce proliferationof the cells. Although not yet definitely confirmed, it is consideredthat the feeder cells function to provide optimal cell density to allowsmall numbers of hybrid cells to multiply more readily. Also, it isthought that the feeder cells may provide the hybrid cells withnutrients required for their proliferation. Various types of feedercells may be employed, including thymocytes from the BALB/c mouse. Othertypes of feeder cells include murine spleen cells, irradiated murineperitoneal exudate cells and murine macrophages. Although variousconcentrations of feeder cells may be added to the culture medium,preferably the feeder cells should be added to the HAT containingcultures at concentrations ranging from 0.5 to 5×10⁶ cells/milliliterwith an optimum density of about 3×10⁶ cells per milliliter.

The different culturing steps of the present invention, discussed above,may be carried out in various environmental conditions. Preferably,however, the cultures should be maintained at a temperature range ofapproximately 35° to 38° C. and in a humidified atmosphere ofapproximately 5 to 10% CO₂ in air. Also, ideally the pH of the culturemedium should be kept in slightly alkaline condition, in a range ofapproximately pH 7.2 to 7.4.

As briefly mentioned above, the present invention also includesidentifying potent sources of anti-IL-2 antibody by cloning hybrid celllines known to produce significant quantities of anti-IL-2 antibody.Thereafter, the cloned hybrid cell lines are cultured in a mediumsupplemented with various additives in substantially the same manner inwhich anti-IL-2 antibodies are produced by hybrid 4E12 cells, asoutlined above. Cloning is accomplished by a limiting dilution procedurewherein the more potent parent hybrid cells, such as 4E12 cells, arecultured in flat-bottomed microplate wells. The hybrid cells areindividually seeded in 200 microliter volumes of fresh Click's mediumsupplemented with 15% by volume FCS, 100 millimolar sodium pyruvate,13.6 milligrams per liter hypoxanthine, 0.176 milligrams per literaminopterin, and 3.88 milligrams per liter of thymidine, (hereaftercollectively referred to as "HAT medium"). Feeder cells, such asthymocytes from the BALB/c mouse in a preferred concentration ofapproximately 3-6×10⁶ cells per milliliter of culture medium, are addedto facilitate cell growth. After approximately one week in culture, themicroplate wells are examined microscopically for cultures containingsingle clusters of hybrid cell growth. Once identified, these growthcultures are fed every three days with 100 microliters of fresh HATmedium. After the hybrid cell populations reach 50 to 70% confluence,the supernate samples produced thereby are assayed for anti-IL-2reactivity.

The clonal cell lines which produce the highest titers of anti-IL-2antibody, such as the hybrid cell line designated in FIG. 1 as 4E12B2,are then subcloned to seek even more potent antibody generating celllines. The technique followed for subcloning is the same as that usedduring the original cloning process. Use of this procedure to subclonethe hybrid 4E12B2 cells has led to the identification of an even morepotent anti-IL-2 antibody producing cell line, designated as 4E12B2D10.As illustrated in FIG. 1, the 4E12B2D10 cell line was found to produceeven greater quantities of anti-IL-2 antibody than generated by the samenumbers of 4E12B2 cells. After potent anti-IL-2 antibody producinghybrid clonal cell lines, such as the 4E12B2D10 cell line, areidentified, they are expanded in HAT-free Click's medium to generatesufficient volumes of supernate to further characterize the antibodycontained in the supernate. Alternatively, the potent anti-IL-2 antibodyproducing hybrid clonal cell lines may be expanded by injecting thecloned anti-IL-2 producing cells into the peritoneal cavities of miceand thereafter collecting the interperitoneal ascites, containing highconcentrations of anti-IL-2 antibody, as explained more fully in Example3 below.

Screening for anti-IL-2 antibody

The anti-IL-2 antibody produced by the hybridoma culture supernates ofthe parent and clonal hybrid cells of the present invention werescreened to ascertain, for example, what cell lines produce significantlevels of antibody. The screening process tested the ability of theantibody to prevent IL-2 dependent replication of effector cellsharvested from long-term culture. The screening process was conductedboth in the presence and in the absence of a complexing agent. Becausehybridoma antibodies are usually incapable of forming largeprecipitating complexes, screening in the absence of a complexing agentwould only detect neutralizing antibodies which are reactive againstdeterminants present on, or near, the active site of IL-2. However, if acomplexing agent is utilized, the screening process, in addition todetecting neutralizing antibodies, would also disclose antibodies thatare directed against determinants elsewhere on the IL-2 molecule.

Briefly, the screening procedure includes suspending approximately 4×10⁴IL-2 dependent CTLL cells in 100 microliter volumes of Click's mediumsupplemented with 10% by volume FCS. Approximately 50 microliters of aone-fifth dilution hybridoma supernate and 50 microliters of rat IL-2(0.5 units per milliliter IL-2 activity) containing conditioned mediumare added to the suspension medium to thereby from 200 microlitervolumes. Thereafter, either 50 microliters of FCS-supplemented Click'smedium (neutralizing screening test) or 50 microliters of lyophilizedStaphylococcus aureus (Igsorb, Enzyme Center, Inc., Boston, Mass.)(precipitating screening tests) are added to the culture. Theprecipitating screening cultures contain a 1/200 final dilution of theIgsorb reagent.

After 24 hours of culturing at 37° C. in a humidified atmosphere of 5%CO₂ in air, the screening cultures are pulsed for approximately 4 hourswith 0.5 microcuries of tritiated thymidine (hereafter "[³ H]Tdr") (NewEngland Nuclear, Boston, Mass.) having a specific activity of 20millicurie per millimole. After pulsing, the cultures are harvested ontoglass fiber filter strips, for instance, with the aid of a multipleautomated sample harvester. [³ H]Tdr incorporation by the CTLL cells ismeasured by liquid scintillation accounting. By this procedure, the CTLLcells which are exposed to IL-2 incorporate significant amounts of(approximately 10,000-20,000 counts per minute) [³ H]Tdr. However, CTLLcells cultured in the presence of IL-2 and anti-IL-2 containinghybridoma supernate incorporates only scintillant control levels of [³H]Tdr (approximately 80-200 counts per minute).

The amount of IL-2 used in screening procedures (approximately 0.5 unitsper culture) was determined by assay of standard IL-2 containingconditioned medium in a CTLL cell replication IL-2 microassay asdiscussed later.

The results of the screening tests are set forth in FIG. 1 above. Of thehybridomas in initial plate #4 (panel A), microwell culture 4E12produced a supernate which significantly inhibited IL-2 dependent CTLLcell [³ H]Tdr incorporation in neutralizing screening tests. Moreover,the addition of .[.Ibsorb.]. .Iadd.Igsorb .Iaddend.in the precipitationscreening culture markedly increased the inhibitory capacity of thesupernate. Subsequent limiting dilution clonings isolated the 4E12B2hybridoma which also markedly inhibited IL-2 dependent proliferation inneutralizing and precipitating screening cultures (FIG. 1, panel B). Thesubcloning of this clonal hybridoma resulted in daughter clones, all ofwhich produced supernates that significantly inhibited IL-2 activity(FIG. 1, panel C, and FIG. 2 below). This indicates that the subclonehybridomas, including the most inhibitory cell line, (4E12B2D10), aremonoclonal.

FIG. 2 shows the results of neutralization (A) and precipitating (B)screening cultures detailing the inhibitory effects (on IL-2 dependentCTLL cell proliferation) of supernates harvested from 4E12B2D10 (),5G8H5C6 () and 2D4A7 () hybrid cell cultures. No inhibition of IL-2induced [³ H]Tdr incorporation was observed in identical screeningassays which tested either HAT medium (Δ) or supernate harvested fromSP2 myeloma cell cultures ( ○ ).

In all screening trials, the control cultures which utilized supernatesharvested from the SP2 myeloma cultures did not mediate any inhibitionof IL-2 induced CTLL cell proliferation, in either the neutralizing orprecipitating screening trials; the SP2 conditioned medium testscreening cultures incorporated from between 8 to 10×10³ counts perminute of [³ H]Tdr. This same result was reached when HAT medium wassubstituted for the .[.hyrbridoma.]. .Iadd.hybridoma .Iaddend.culturesupernates in control experiments.

Fractionation of Anti-IL-2 Activity of Protein A Affinity ColumnChromatography

As discussed above, the observation that all 4E12B2 daughter clonedcells produced supernates which significantly inhibited IL-2 activitypoints to the monoclonality of these hybridomas. Further studiesdemonstrated that the anti-IL-2 activity present in the hybridomasupernates had a high affinity for protein A. Since it is known thatprotein A has a high affinity for the Fc portion of immunoglobulin Gmolecules, the affinity of the anti-IL-2 activity for protein A suggeststhat the anti-IL-2 inhibitory activity present in 4E12B2D10 hybridomasupernates is due to the presence of .[.a.]. .Iadd.an .Iaddend.IgGantibody. As discussed above, the IgG was the product of single clones.

The procedure for fractionating the hybridoma supernates and thentesting the inhibitory effect of the fractions on IL-2 activity.[.,.].included precipitating protein present in 25 milliliters of 24 hoursupernates harvested from 4E12B2D10 hybridoma cultures by slowly addingsolid ammonium sulfate until a final saturation of 70% (weight/volume)was achieved. After overnight stirring of the mixture at 4° C., theprotein precipitate was pelleted by a 20 minute centrifugation at10,000× g. The resulting protein pellets were resuspended in as small avolume of sterile 0.9% NaCl (pH 7.2) as possible and then dialyzed for24 hours against 1000 volumes of 0.9% NaCl. Three milliliters of theresulting dialyzed protein solution were applied to a column of ProteinA Sepharose (Pharmacia Fine Chemicals, Piscataway, N.J.). The Sepharosecolumn had been previously equilibrated in 0.9% NaCl (pH 7.2) andsubsequently washed with additional 0.9% NaCl. The filtrate remainingfrom the Sepharose column was retained and tested for anti-IL-2activity. To elute the hybridoma product which bound to the Protein ASepharose matrix, the column was washed with 0.2 molar glycine HCl (pH3)buffer. The column acid wash (eluate) was then dialyzed against 100volumes of 0.9 NaCl (pH 7.2) and followed by dialysis against 100volumes of Hank's buffered salt solution (hereafter "HBSS").

Thereafter, the eluate was tested for anti-IL-2 activity by use of theabove-described precipitating screening culture. The filtrate remainingfrom the application of the hybridoma to the Protein A Sepharose columnwas also tested. As set forth in FIG. 3 below, the acid eluate solutionexhibited pronounced anti-IL-2 activity whereas the filtrate did not.FIG. 3 also charts the capacity of the crude 4E12B2D10 supernate toinhibit IL-2 activity, as measured by its capacity to inhibit IL-2dependent CTLL cell [³ H]Tdr incorporation. As in FIG. 2 above, FIG. 3illustrates that both HAT medium and supernate harvested from SP2myeloma cell culture had no capacity to inhibit IL-2 activity inscreening assays.

In summary, the data set forth in FIG. 3 suggests that the anti-IL-2inhibitory activity present in the 4E12B2D10 hybridoma supernate is dueto the presence of a monoclonal (IgG) antibody which can be bound andeluted from a Protein A Sepharose column. Further, subtyping studiesrevealed that monoclonal anti-IL-2 was an IgG of the gamma 2_(b)subclass.

FIG. 3 shows Igsorb-precipitation screening cultures monitoring theanti-IL-2 effects of HAT medium ( ○ ), SP2 conditioned medium () andsupernate harvested from the cloned hybridoma 4E12B2D10 cell line (.[..]..Iadd. .Iaddend.). The capacity of the 4E12B2D10 supernate to inhibitIL-2 dependent CTLL cell [³ H]Tdr incorporation was lost by passage ofthe supernate over a 3 ml column of Protein A conjugated Sepharose(.[.□.]..Iadd. .Iaddend.). However, enhanced anti-IL-2 inhibitoryactivity could be recovered from the column by standard acid elutionusing glycine-HCl (pH3) buffer and subsequent dialysis (□).

Inhibition of rat, mouse and human IL-2 activity by purified 4E12B2D10hybridoma antibody

By use of the above-described Igsorb precipitation tests, applicant hasfound that the 4E12B2D10 antibody, purified by the previously discussedProtein A Sepharose affinity procedure, has the ability to inhibit,equally effectively, rat, mouse and human IL-2 activity. As detailed inTable I below, two other hybridoma supernates, denominated as 5GBH5C6and 2D4A7, exhibited this same ability. These results suggest that theantibodies isolated from the hybridoma supernates by Protein A Sepharosepurification react with a determinant shared by all three speciesclasses of IL-2.

                                      TABLE I                                     __________________________________________________________________________    Capacity of Hybridoma Antibody to Inhibit Mouse, Rat and Human IL-2           Activity in Precipitation Assays                                                       Reciprocal of Supernate Dilution                                              Mouse IL-2        Rat IL-2    Human IL-2                                      1/10    1/20                                                                              1/40  1/10                                                                              1/20                                                                              1/40                                                                              1/10 1/20 1/40                         __________________________________________________________________________    HAT Medium                                                                             10.3.sup.(a) (-11).sup.(b)                                                            9.4(-1)                                                                            9.0(4)                                                                             9.3(0)                                                                            9.2(1)                                                                            8.9(4)                                                                            9.9(-4)                                                                            9.2(1)                                                                             10.6(-13)                    SP-2 conditioned                                                                       9.1(4)  9.5(-2)                                                                            9.9(-4)                                                                            9.2(1)                                                                            9.3(0)                                                                            9.3(0)                                                                            9.8(-8)                                                                            9.6(-3)                                                                            9.7(-4)                      medium                                                                        4E12B2D10                                                                              1.2(81) 2.7(71)                                                                            4.6(51)                                                                            0.7(93)                                                                           2.8(76)                                                                           5.5(41)                                                                           0.8(93)                                                                            3.2(66)                                                                            6.1(35)                      Protein A-                                                                    Sepharose eluate                                                              5G8H5C6  0.8(92) 2.0(79)                                                                            4.2(55)                                                                            0.5(95)                                                                           1.9(80)                                                                           6.0(36)                                                                           2.6(72)                                                                            3.2(66)                                                                            7.9(16)                      Protein A-                                                                    Sepharose eluate                                                              2D4A7    0.3(97) 1.5(84)                                                                            3.5(63)                                                                            2.8(70)                                                                           3.0(68)                                                                           4.9(48)                                                                           1.3(87)                                                                            4.3(54)                                                                            5.6(40)                      Protein A-                                                                    Sepharose eluate                                                              __________________________________________________________________________     .sup.(a) cpm × 10.sup.-3 CTLL.sup.3 HTdr incorporation.                 .sup.(b) % inhibited of counted proliferation (9.3 × 10.sup.3 cpm o     .sup.3 HTdr incorporated by 4,000 CTLL cells cultured in the presence of      50% concentration of 1 U/ml rat IL2).                                    

The Igsorb precipitation procedure was essentially the same as that usedduring anti-IL-2 antibody screening protocols, as described above. Eachof the culture samples contained approximately 4000 CTLL cells, 0.5units per milliliter of IL-2 (regardless of the species source), adiluted quantity of eluted antibody and a 1/200 final dilution ofIgsorb.

Rat IL-2 containing conditioned medium was prepared in the same manneras the IL-2 used to immunize the BALB/c female mice prior tohybridization, as set forth above. Mouse IL-2 containing conditionedmedium was prepared by PHA (1% by volume, Grand Island Biologicals,Grand Island, N.Y.) stimulation of 10⁶ LBRM-33 mouse lymphoma cells permilliliter of conditioned medium. Human IL-2 containing conditionedmedium was prepared by identical mitogen stimulation of (i) normal humansplenocytes wherein 10⁷ splenocyte cells per milliliter of culturemedium were utilized, or (ii) Jurkat-FHCRC human leukemic T cellswherein 10⁶ malignant cells per milliliter of culture medium wereutilized.

Immune precipitation of IL-2 biological activity

Applicant has ascertained that the 4E12B2D10 derived antibody, IgG,after being removed from hybridoma supernate by the above-describedProtein A Sepharose procedure, was capable of precipitating IL-2activity. This finding supports the conclusion that the 4E12B2D10culture consists of monoclonal B cells whose immunoglobulin G productappears to be directed against a determinant present on molecules ofhuman, murine and rat IL-2.

In the precipitation procedure, individual 0.5 milliliter aliquots of:(i) SP2 conditioned medium; (ii) HAT medium, and (iii) a 1/20 dilutionof Protein A Sepharose purified 4E12B2D10 IgG, were mixed in equal 0.5volumes of rat conditioned medium containing 3.0 units per milliliter ofIL-2. The mixtures were incubated in 15×75 millimeter glass centrifugetubes for 30 minutes at 37° C. Thereafter 250 microliters of Igsorb(1/16 dilution in Click's medium) was added to each centrifuge tube andthe incubation continued for an additional 45 minutes. After theincubation was completed, each sample was centrifuged for 20 minutes at400×g and then the supernate samples assayed for residual IL-2 activityin standard IL-2 dependent CTLL [³ H]Tdr incorporation assays, asdiscussed below.

The results of the tests are set forth in FIG. 4 below. Only the testtrials involving incubation of IL-2 activity with 4E12B2D10 antibodyresulted in immune precipitation of rat IL-2 biologic activity. Similarpretreatment of rat IL-2 conditioned medium with equal volumes of either(i) medium conditioned by SP2 myeloma cells or (ii) HAT medium resultedin diminution of IL-2 activity titers corresponding only to the dilutioncaused by such addition.

FIG. 4 shows the results of CTLL proliferation assays testing IL-2activity remaining in standard preparation of rat spleen cellconditioned medium (3 U/ml) either before () or following Igsorbprecipitation in the presence of Click's medium (Δ), SP2 conditionedmedium ( ○ ), HAT medium () or Protein A Sepharose purified.[.4E12B2D101gG.]. .Iadd.4E12B2D10 IgG .Iaddend.(). Only immuneprecipitation with 4E12B2D10 antibody resulted in a significantdiminution of IL-2 activity beyond that demonstrated by dilution.

Immune precipitation of radio-labeled IL-2 activity

Applicant has determined that the 4E12B2D10 hybridoma antibody also hasthe ability to precipitate radio-labeled IL-2. This result not onlyconfirms that the 4E12B2D10 supernate contains monoclonal B cellhybridomas having antibody products which are reactive with determinantspresent on the IL-2 molecule, but also provides the bases for a processby which the presence of IL-2 may be detected with less effort thanrequired by conventional assay processes.

Prior to the actual precipitation process, biosynthetically ³ H-labeledIL-2 activity was prepared by culturing the IL-2 producing murine tumorcell line, LBRM-33, in the presence of five different amino acids, eachbearing a ³ H-label, i.e. leucine, lysine, phenylalanine, proline andtyrosine (Catalog No. TRK550, Amersham Corp., Arlington Heights, Ill.).In the ³ H-labeled IL-2 production process, 7×10⁵ LBRM-33 cells permilliliter of culture volume were cultured in 25 milliliter volumes ofRPMI 1640 medium supplemented with 2% by volume FCS. Each culture samplecontained a deficient (20% of normal) concentration of each of the fiveamino acids. Also, at the beginning of the culture period, an additionalamount of each particular amino acid, which previously had been tritiumlabeled, was added to the corresponding culture to achieve a finalcomposite concentration of 10 microcuries per milliliter. After 48 hoursof culture, the T cell plant mitogen, PHA, was added to each of thecultures in a final concentration of 1% by volume. Twenty-four hourslater, the IL-2 containing supernates were harvested and thenconcentrated by 85% ammonium sulfate precipitation. Thereafter, thesupernates were fractionated by sequential diethyl amino .[.ethly.]..Iadd.ethyl .Iaddend.(hereafter "DEAE") cellulose ion exchangechromatography and Sephadex G-100 gel exclusion chromatography aspreviously detailed in Gillis et al, "Biochemical and BiologicalCharacterization of .[.Lympocytes.]. .Iadd.Lymphocytes.Iaddend.Regulatory Molecules-II. Purification of a Class of Rat andHuman Lymphokines", 124 The Journal of Immunology 1954 (1980). Thisprocess resulted in production of active fractions (20 to 100 units permilliliter) in the 33,000 to 36,000 molecular weight range, eachcontaining considerable radio activity (approximately 100,000 counts perminute/milliliter) (see FIG. 5 below).

FIG. 5 shows IL-2 activity () and cpm of ³ H-label () in Sephadex.[.G100.]. .Iadd.G-100 .Iaddend.fractions of biosyntheticallyradio-labeled LBRM-33 cell derived IL-2 preparations. Prior to gelexclusion chromatography, radio-labeled IL-2 present in LBRM-33conditioned medium was concentrated by ammonium sulfate precipitationand DEAE ion exchange chromatography.

In the immune precipitation process itself, the ³ H-labeled IL-2containing fractions were diluted in Click's medium so that 20microliter aliquots of the fractions each contained radioactivity levelsof approximately 1200 counts per minute. The 20 milliliter aliquots wereincubated at 37° C. in the presence of serial log₂ dilutions ofhybridoma supernate. Each supernate sample was placed in a sterile 15×75milliliter glass centrifuge tube along with 10% by volumeFCS-supplemented Click's medium collectively to compose 50 microlitervolumes. The mixtures were incubated for 30 minutes at 37° C. in anatmosphere of 5% CO₂ in air. Thereafter, 20 microliters of Igsorb (1/2dilution) .[.was.]. .Iadd.were.Iaddend.added to each tube and theincubation continued for an additional 45 minutes. Then, 2 millilitersof 0.9% NaCl .[.was.]. .Iadd.were .Iaddend.added to each tube and theimmune precipitate pelleted by centrifuging for 5 minutes at 400×g. Thepellet was washed twice in 2 milliliter volumes of 0.9% NaCl. After thefinal wash, 3.5 milliliters of Bioflour scintillation cocktail (NewEngland Nuclear) .[.was.]. .Iadd.were .Iaddend.added to each tube andthe contents vortexed, prior to being transferred to plastic miniscintillation vials. Lastly, the tritium radioactive label associatedwith the pellet was counted by liquid scintillation. The countingresults of the immune precipitation of ³ H-labeled IL-2 activity are setforth in Table II, below.

                  TABLE II                                                        ______________________________________                                        Immune Precipitation of Radiolabeled IL-2 Activity.                                          CPM of .sup.3 H-IL-2 Associated                                               with Igsorb Pellet.sup.1                                                      Supernate Dilution                                             Culture Supernate                                                                              1/5    1/10   1/20  1/40 1/80                                ______________________________________                                        HAT medium        80    110     75    95   86                                 SP-2             123    116    135   117  110                                 4E12B2D10        856    737    794   544  244                                 4E12B2D10 + XC Cold-IL-2                                                                       172    134    136   121  129                                 4E12B2D10 Protein A                                                                            186    245    615   775  892                                 Sepharose-Acid Eluate                                                         ______________________________________                                         .sup.1 1200 cpm added to each reaction mixture                           

The same precipitation test was conducted using log₂ serial samples ofHAT medium, SP2 conditioned medium and a 1/5th dilution of the 4E12B2D10hybridoma supernate, see Table II. It is interesting to note that thecapacity of the 4E12B2D10 supernate to precipitate radio-labeled IL-2remained at a high 794 count per minute level even when a 1/20 dilutionof the hybridoma supernate was tested, and could be inhibited byperformance of an identical immune precipitation in the presence of a10-fold excess concentration of unradiolabeled IL-2.

4E12B2D10 IgG Affinity Chromatography

The conclusion that the 4E12B2D10 hybridoma cell line produces amonoclonal IgG antibody directed against some determinant present on theIL-2 molecule was further confirmed through tests which found thatCNBr-mediated coupling of the hybridoma antibody to Sepharose 4Bresulted in the preparation of a matrix capable of absorbing significantquantities of IL-2. This result indicates that the antibody generated bythe present invention may be useful in affinity purification ofbiologically active IL-2.

The test procedure utilized included purifying by Protein A Sepharoseaffinity chromatography (as outlined above) concentrated hybridomasupernate from the 4E12B2D10 hybrid cell line. The antibody eluted fromthe Protein A Sepharose column chromatography purification procedure wasdiluted to a concentration of approximately 2 milligrams per milliliterin a coupling buffer composed of 0.5 molar NaCl and 0.1 molar NaHCO₃ (pH8.3). The approximate antibody concentration was determined byultraviolet light absorption (280 nM) using a Beckman Spectralphotometer (using the assumption that .[.E₂₈₀ ¹ .]..Iadd.E'280.Iaddend.=10.0).

Prior to the addition of the purified antibody, one gram of the CNBractivated Sepharose 4B (Pharmacia Fine Chemicals.[.,.]. was swollen bywashing with 200 milliliters of cold 1 millimolar HCl. The swollenSepharose 4B was pelleted by centrifuging for 10 minutes at 200×g. Next,the activated CNBr Sepharose 4B was washed with 0.9 NaCl, resuspended incoupling buffer to form a 3 milliliter slurry and then the slurry mixedwith 1 milliliter of 4E12B2D10 antibody in a 15 cubic centimeter conicalcentrifuge tube.

This was thereafter placed on an end over end shaker for 2 hours at roomtemperature to promote coupling of the antibody to the CNBr Sepharose4B. Thereafter, the gel was pelleted and the excess unbound antibodyremoved. The CNBr active antibody sites which were left uncoupled by theabove coupling process were blocked by resuspending the gel in 100milliliters of 0.5 molar NaCl and 0.2 molar glycine (pH 8.0) buffer. Thegel was again mixed for 2 hours on an end over end shaker.

As an additional control to ensure that any IL-2 activity removed byantibody-coupled Sepharose could not be due to direct coupling of theIL-2 to CNBr active Sepharose sites, a second CNBr Sepharose slurry wasprepared. A 1 milliliter solution of 2 milligrams per milliliter ofbovin serum albumin (hereafter "BSA") was coupled to this second slurryusing the same coupling procedure as set forth above.

Both IgG and BSA coupled Sepharose gels, following treatment with 0.5molar NaCl, 0.2 molar glycine (pH 8.0) buffer, were washed sequentiallywith three 10 milliliter volumes of coupling buffer, three 10 millilitervolumes of 0.1 molar sodium acetate buffer (in 0.5 molar NaCl, pH 4) andthree additional 10 milliliter volumes of coupling buffer. Prior to usein IL-2 affinity .[.chromotography.]. .Iadd.chromatography.Iaddend.trials, the columns equilibrated in sterile 0.9% NaCl (pH 7.2).

In the affinity trials, 10 milliliters of rat spleen cell conditionedmedium, having an IL-2 activity level of 3 units per milliliter, werepassed over each gel column. The resulting filtrates were collected andtested for residual IL-2 activity in standard IL-2 microassays, which asdetailed below, monitored the IL-2 dependent [³ H]Tdr incorporation ofCTLL cells. As set forth in FIG. 6 below, the filtrate collected frompassage of the rat spleen cell conditioned medium over theBSA-conjugated Sepharose column contained as much IL-2 activity as thatpresent in the original rat spleen cell-derived IL-2 solution. On theother hand, the filtrate collected after a single passage of the ratspleen cell conditioned medium over the column of 4E12B2D10antibody-coupled Sepharose contained less than 5% of the IL-2 activitypresent in the original conditioned medium. The ability of anti-IL-2antibody coupled to CNBr-Sepharose to absorb IL-2 activity further.[.cnfirms.]. .Iadd.confirms .Iaddend.that a monoclonal antibodydirected against some determinate present on the IL-2 molecule isproduced by the 4E12B2D10 hybridoma cell line.

FIG. 6 shows IL-2 activity present in an 3 U/ml .[.sammple.]..Iadd.sample .Iaddend.of rat spleen cell conditioned medium both priorto () and following passage over a BSA conjugated Sepharose column () oran identical column to which .[.4E12B2D101gG.]. .Iadd.4E12B2D10 IgG.Iaddend.had been coupled ().

Standard IL-2 Microassay

The IL-2 concentration in the above-described immune precipitation,affinity .[.chromotography.]. .Iadd.chromatography.Iaddend., andscreening experiments (testing the effectiveness of the anti-IL-2antibody produced by the parent and clonal cell lines) was determined bya conventional IL-2 microassay as outlined in Gillis et al. "T-CellGrowth Factor: Parameters of Production and a Quantitative Microassayfor Activity", 120 The Journal of Immunology, 2027 (1978). Briefly, theassay procedure includes seeding approximately 4000 murine CTLL cells ina log₂ dilution series of the given IL-2 sample. In each culture, theCTLL cells .[.ae.]. .Iadd.are .Iaddend.suspended in Click's mediumsupplemented with 10% by volume FCS to thereby form 200 microliter totalvolumes. The cultures are incubated for approximately 12 hours at 37° C.in a humidified atmosphere of 5% CO₂ in air. Thereafter, the culturesare pulsed for approximately 4 hours with 0.5% microcurie of [³ H]Tdrhaving a specific activity of 20 microcurie per millimolar (obtainedfrom New England Nuclear, Boston, Mass.).

After pulsing, the cultures are harvested onto glass fiber filterstrips, for instance with the aid of a multiple automated sampleharvested. [³ H]Tdr incorporation by the CTLL cells .[.as.]. .Iadd.is.Iaddend.measured by liquid scintillation counting. By this procedure,the CTLL cells which are cultured in the presence of IL-2 willincorporate [³ H]Tdr in a dose dependent manner. On the other hand, CTLLcells cultured in the absence of IL-2 will incorporate only scintillantcontrol levels of [³ H]Tdr and will be more than 95% trypan-bluepositive after 24 hours of IL-2 deprivation, indicating that such cellsare dead.

The concentration of IL-2 is quantified by probix analysis of thymidineincorporation data. A 1 unit per milliliter standard of IL-2 activity isdefined as the amount of IL-2 activity present in a 48 .[.hours.]..Iadd.hour .Iaddend.tissue culture medium conditioned by Con A (5micrograms/milliliter) stimulation of an initial concentration of 10⁶normal rat spleen cells per milliliter of culture. A 1 unit permilliliter standard of IL-2 activity routinely stimulated approximately10,000 counts per minute of [³ H]Tdr incorporation at a dilution of 1:2.

EXAMPLE 1

Six week old BALB/c female mice were immunized once weekly for fourweeks with 3000 units of rat IL-2. The IL-2 used for the immunizationprogram was prepared by stimulating a concentration of 1×10⁷ ratsplenocyte cells per milliliter of culture medium for 24 hours with 5micrograms of Con A per milliliter of culture medium. The supernateproduced thereby was purified sequentially by ammonium sulfateprecipitation, Sephadex G-100 gel exclusion .[.chromotography.]..Iadd.chromatography.Iaddend., DEAE cellulose ion exchange.[.chromotography.]. .Iadd.chromatography .Iaddend.and preparativeflat-bed iso-electric focusing, as described in Gillis et al., supra,124 The Journal of Immunology 1954 (1980).

During each immunization, 1000 units of IL-2 in 0.2 milliliter volumes.[.was.]. .Iadd.were .Iaddend.injected intradermally in each hind leg.Prior to the first immunization the IL-2 was mixed with completeFreund's adjuvant, and prior to the last three immunizations, the IL-2was mixed with incomplete Freund's adjuvant. Also, during eachimmunization, 1000 units of rat IL-2, in a volume of 0.5 milliliters of0.9% NaCl, .[.was.]. .Iadd.were .Iaddend.injected intraperitoneally.

Two days prior to fusion, spleens from the IL-2 immunized mice wereremoved and single cell suspensions prepared therefrom. The splenocyteswere cultured for 48 hours in Click's medium. The medium wassupplemented with 10% by volume, heat-inactivated (56° C. for thirtyminutes) FCS, 25 mM HEPES buffer, 16 mM NaHCO₃, 50 micrograms permilliliter of streptomycin, 50 units per milliliter penicillin and 300micrograms per milliliter of fresh L-glutamine. The splenocytes wereactivated by adding 100 micrograms of E. Coli lipopolysaccharide permilliliter of culture. The culture was maintained at approximately 37°C. in a humidified atmosphere of 5% CO₂ in air.

Fusion was achieved by mixing approximately 8×10⁷ of the LPS-activated"IL-2 immune" splenocytes with approximately 2×10⁷ SP2 myeloma cellsobtained from the BALB/c mouse. This cell line is a derivative of theSP2/HLGK myeloma line which does not constitutively produceimmunoglobulin light chain. The cell mixture was pelleted bycentrifuging for five minutes at 200×g. The resulting pellet was thenimmersed in a 37° C. water bath and carefully resuspended over a periodof one minute with one milliliter of warm Click's medium (.[.PH.]..Iadd.pH .Iaddend.7.3 and without FCS) containing 40% (weight/volume)polyethylene glycol 1500. Two more milliliters of warm, serum-freeClick's medium were then added over the next two minute period.Thereafter, eight additional milliliters of this medium were added.Next, the mixture was centrifuged for five minutes at 200×g to completethe fusion process.

Constitutive production of anti-IL-2 antibody derived from the fusion ofactivated, IL-2 immunized, BALB/c splenocytes with SP2 myeloma cells wasachieved by suspending the resulting cell pellet in 40 milliliters ofClick's medium supplemented with 15% by volume FCS and 100 millimolarsodium pyruvate. The unfused myeloma driver cells were prevented fromproliferating by the addition to the medium of 13.6 micrograms per literof hypoxanthine, 0.176 micrograms per liter of aminopterin and 3.88micrograms per liter of thymidine. The fused cell solution was thenadded to another 160 milliliters of the same medium which also containedapproximately 8×10⁸ BALB/c female mice thymocyte cells to serve asfeeder cells. The entire cell suspension was gently mixed and thendivided into 200 microliter aliquots in flat-bottomed microplate wells(No. 3596 Costar, Inc., Data Packaging, Cambridge, Mass.). The cultureswere all maintained in approximately 37° C. in a humidified atmosphereof 5% carbon dioxide in air.

EXAMPLE 2

Anti-IL-2 antibody was constitutively produced by culturingapproximately 10⁶ 4E12B2D10 clonal hybrid cells per milliliter ofculture medium in either 2 milliliter volumes in cluster plates (No.3524, Costar, Inc., Cambridge, Mass.) or in 10 milliliter volumes inculture flasks (No. 3013, Falcon Plastics, Oxnard, Calif.). The culturemedia contained Click's medium supplemented with 15% by volume, heatinactivated (56° C. for 30 minutes) FCS and 100 mM sodium pyruvate. Theculture was maintained at approximately 37° C. in a humidifiedatmosphere of 5% CO₂ in air. Supernate harvested after 24 hours ofculture contained approximately 5-15 micrograms/milliliter of anti-IL-2Immunoglobulin G.

EXAMPLE 3

Anti-IL-2 antibody was produced in high concentration in vivo byintraperitoneal injection of BALB/c female mice with 10⁶ 4E12B2D10hybridoma cells. One week prior to hybridoma cell injection, BALB/c micewere given 0.5 milliliter of pristane .[.intraperioneally.]..Iadd.intraperitoneally .Iaddend.as an ascites inducing irritant. From14 to 17 days after hybridoma injection intraperitoneal ascites.[.was.]. .Iadd.were .Iaddend.collected, and the anti-IL-2Immunoglobulin G contained therein was purified by its affinity toProtein A-Sepharose.

As will be apparent to those skilled in the art to which the inventionis addressed, the present invention may be carried out by using murinemyeloma cell lines, culture media, culture media additives, T and B cellstimulants, fusing .[.atents.]. .Iadd.agents .Iaddend.and cell growthinhibitors specifically disclosed above without departing from thespirit or essential characteristics of the invention. The particularprocesses described above are therefore to be considered in all respectsas illustrative and not restrictive, i.e..Iadd., .Iaddend.the scope ofthe present invention is as set forth in the appended claims rather thanbeing limited to the examples of the anti-IL-2 antibody producingprocesses as set forth in the foregoing description.

What is claimed is:
 1. An anti-IL-2 antibody produced by:immunizingmurine B-lymphocyte cells with IL-2; activating said immunized murineB-lymphocyte cells with a B-cell mitogen; fusing said activated murineB-lymphocyte cells with murine myeloma cells; .[.culturing.]..Iadd.cultivating .Iaddend.said hybridoma anti-IL-2 antibody producingcells under conditions suitable for antibody production; and recoveringthe anti-IL-2 antibody produced by said anti-IL-2 antibody producingcells.
 2. The anti-IL-2 antibody of claim 1, wherein said B-lymphocytecells are from the BALB/c mice. .[.3. The anti-IL-2 antibody of claim 1,wherein said hybridoma anti-IL-2 antibody producing cells include cellsselected from the group consisting essentially of 4E12, 4E12B2 and4E12B2D10 cells..]. .Iadd.4. A monoclonal antibody that specificallybinds IL-2, wherein said antibody is produced by a murine hybridomawhich is the fusion product of a murine B lymphocyte and a murinemyeloma cell..Iaddend. .Iadd.5. A monoclonal antibody according to claim4, wherein said antibody inhibits T-cell proliferation in the presenceof interleukin-2. .Iaddend. .Iadd.6. A monoclonal antibody according toclaim 4, wherein said antibody immunoprecipitates interleukin-2molecules. .Iaddend. .Iadd.7. A monoclonal antibody produced by a cellline selected from the group consisting of ATCC HB8093, ATCC HB8092 andATCC HB8091, and which specifically binds to IL-2. .Iaddend.